All-trans-retinoic acid (atRA), the biologically active metabolite of dietary Vitamin A, is essential for mediating diverse biological functions in multipl tissues such as the liver, kidney, lung and pancreas. The different biological actions of atRA are regulated through tissue concentration gradients of atRA, but there are considerable gaps in our knowledge on how the tissue specific signaling of atRA is regulated during childhood and adult life. We propose that atRA concentration gradients are generated by regulated expression and activity of the enzymes synthesizing atRA (ALDH1As), those that metabolize atRA (CYP26s) and cellular retinoic acid binding proteins (CRABPs). Our central hypothesis is that alterations in the activity or expression of these enzymes change atRA signaling and distribution, contribute to disease development in specific tissues and result in adverse effects. To test this hypothesis we will first characterize atRA metabolism in cell systems and establish the role of cellular retinoic acid binding proteins (CRABPs) in modulating atRA clearance, signaling and distribution. We will use basic biochemical and enzyme kinetic methods and in vitro cell experiments to establish the role and kinetics of direct protein-protein interactions between CYP26s and CRABPs. We will then establish the tissue and cell type specific roles of ALDH1A, CYP26 and CRABP enzymes in maintaining atRA homeostasis in humans and mice. This will be done using normal human and mouse tissues, novel high sensitivity mass spectrometry methods, generating new conditional knock-out mice of CYP26 enzymes, testing pharmacological effects of CYP26 and ALDH1A inhibitors and using physiologically based pharmacokinetic (PBPK) modeling. To determine the overall physiological importance of these enzymatic processes, we will use the knock-out mice and our pharmacological tools in vitro and in vivo, to demonstrate that altered CYP26 or ALDH1A activity impairs normal physiological atRA signaling in target tissues. We will focus on atRA signaling in the liver, kidney, pancreas and lung due to the existing knowledge that atRA signaling plays a fundamental role in these tissues. Together these studies will generate a new and unique integrative model of retinoid homeostasis that will be useful in evaluating and predicting the effects of xenobiotics, new therapeutic approaches, disease processes and genetic factors in altering tissue retinoid signaling. This will have major impact in improving human health as it has direct application in improving our understanding of the processes involved in lipid and glucose homeostasis in the liver and the pancreas, in development and treatment of nephropathies and in lung alveoli health. In addition, the knowledge gained through these studies will improve our understanding of the role of atRA signaling during childhood development and maturation, and can be extended to improve our understanding of the role of atRA in skin diseases such as psoriasis and ichthyosis and in neurodegenerative diseases such as Alzheimer's and dementia in which atRA signaling has been shown to be altered.